We have used gene targeted mutation techniques in ES cells to demonstrate that the I region is a critical element in the control of Ig heavy chain class switch recombination (CSR), to show that transcription per se through the S region is not sufficient to target efficient CSR, and to generate evidence for the presence of a long range CSR control region associated with the 3' lg heavy chain enhancer (3'EH) at the 3' end of the IgH locus. We now propose to continue to use ES cell, somatic chimera, and germline mutation technologies to elucidate in detail the molecular elements that control CSR. An initial focus will be to target l region mutations into one IgH allele of an Fl ES cell line which carries allotypic markers that distinguish allelic CH genes and products and to use these mutant cells in the RAG-2 deficient blastocyst system to rapidly assay switching on targeted and non-targeted chromosomes. Mutations with novel phenotypes also will be targeted into the J-1 ES cell line and introduced into the mouse germline for further analyses. We will use these approaches to generate and assay specific mutations, via the LoxP/Cre recombinase system, of the I-gamma2b and/or 1-epsilon exons to test the role of I regions, I region promoter sequences, as well as primary and processed germline CH transcripts in targeting CSR. We will also continue to elucidate the mechanism by which the previously characterized 3'ER replacement mutation affects CSR and germline CH transcription. A major goal will be to test whether this mutation exerts its effects through deletion of a critical element (e.g. the 3'EH sequences), insertion of a competing promoter, or both. For this purpose, we will compare the germline CH transcription and CSR ability of B cells derived from ES cells that either harbor an inserted neo-r gene upstream or in place of the 3' EH in F1 ES cells to that of B cells derived from ES cells in which the net gene was deleted via the loxP/Cre system. Finally, we will generate additional deletion and/or replacement mutations of other DNA regions upstream or downstream of the 3'EH (e.g. the C-alpha3'EH and putative C- gamma1 enhancer, plus DNA sequences encompassing all or a subset of 4 DNAse hypersensitivity sites within and downstream of the 3'EH region) to assay for additional controlling sequences with respect to germ line CH transcription and Ig heavy chain class switching.